Abstract

Background: TNBC or basal-like subtype of breast cancer confers poor clinical outcome mostly due to its inherent aggressive nature and its high frequency of metastasis. Malignancy in TNBC is associatedwith micro-vascular proliferations. Vascular Mimicry (VM) of solid tumor is an endothelium-independent matrix-embedded, blood-perfused non-angiogenic micro-circulatory phenomenon. VM of tumor cells refer to the characteristic plasticity of aggressive cancer cells forming de novo vascular networks which perfuse rapidly growing tumors, transporting fluid from leaky vessels and/or connect with the constitutional endothelial-lined vasculature. In a study comparing the VM in three subsets of BC, the presence of VM was more profoundly observed in TNBC than in luminal and HER2+ subtypes.

Hypothesis: Recently we have identified a differential activation of Wnt-b-Catenin pathway (WP) in TNBC (Dey et al., PLOSONE; 2014) and demonstrated that Wnt signaling in TNBC is association with metastasis (Dey et al., BMC Cancer 2013). Since TNBC is an aggressive form of BC and VM is associated with the aggressiveness / poor outcome in many solid tumors including BC, we hypothesized that the upregulation of WP in TNBC may have a functional relationship with VM.

Methods: Four strategies were used to study the role of WP in VM. They are (1) Wnt-receptor interaction by using WntC59, (2) Wnt cytosolic signaling by using XAV939, (3) Wnt nuclear signaling by using sulindac sulfide and beta-catenin SiRNA and (4) Wnt-regulating pathway by using pan-PI3K inhibitor (LY294002 and SF1126). The signaling of VM was characterized by bevacizumab (HUVEC cell cord formation used as positive control).

Results: Using pharmacological and genetic tools, we demonstrated that perturbation of WP (1) at its different nodes and (2) via its upstream regulatory pathway (PI3K) can regulate VM in TNBC cells. Data showed that (1) sulindac sulfide, (2) XAV939 and WntC59 and (3) LY294002 or SF1126 (which decreased pGSK3beta) caused an abrogation of VM in TNBC cell lines while bevacizumab failed to affect the vessel formation. Considering the involvement of VM in mediating the aggressive/metastatic nature of TNBC, we finally tested the effect of downregulation of WP on VM in brain metastasis specific EGFP-tagged MDA-MB231BR cells using a real time recording device (Incucyte) to demonstrate that VM in these cells is regulated by WP.

Significance: To our knowledge, this is the first report to demonstrate a direct functional role of WP in the regulation of VM in TNBC.